Tonantzitlolone B Modulates the Endogenous Opioid System to Promote Antinociception in Mice.

Tonantzitlolone B (TZL-B) is a diterpene isolated from the roots of Stillingia loranthacea. Its antinociceptive effects were investigated in male Swiss mice using the following models of pain: formalin test, inflammation induced by Complete Freund's Adjuvant (CFA), tail flick test, and cold plate test. The influence of TZL-B on the opioid system was assessed in vivo, using opioid antagonists; in silico, investigating the chemical similarity among TZL-B and opioid agonists; and ex vivo, measuring preproenkephalin (PENK) gene expression in the spinal cord by RT-qPCR. TZL-B (10-1000 µg/kg) promoted antinociception in the four experimental models without impairing mice's motor function. TZL-B did not alter paw edema during CFA-induced inflammation. The antinociceptive effects of TZL-B in the tail flick and cold plate tests were diminished by the opioid antagonists naloxone (5 mg/kg), NOR-BNI (0.5 mg/kg), naltrindole (3 mg/kg), and CTOP (1 mg/kg), indicating the involvement of κ-, δ-, and µ-opioid receptors. TZL-B showed no significant chemical similarity to opioid agonists, but the treatment with TZL-B (1000 µg/kg) increased PENK gene expression in the spinal cord of mice. These data suggest that TZL-B promotes antinociception by enhancing the transcription of PENK, hence modulating the endogenous opioid system.

Unveiling Targets for Treating Postoperative Pain: The Role of the TNF-α/p38 MAPK/NF-κB/Nav1.8 and Nav1.9 Pathways in the Mouse Model of Incisional Pain.

Although the mouse model of incisional pain is broadly used, the mechanisms underlying plantar incision-induced nociception are not fully understood. This work investigates the role of Nav1.8 and Nav1.9 sodium channels in nociceptive sensitization following plantar incision in mice and the signaling pathway modulating these channels. A surgical incision was made in the plantar hind paw of male Swiss mice. Nociceptive thresholds were assessed by von Frey filaments. Gene expression of Nav1.8, Nav1.9, TNF-α, and COX-2 was evaluated by Real-Time PCR in dorsal root ganglia (DRG). Knockdown mice for Nav1.8 and Nav1.9 were produced by antisense oligodeoxynucleotides intrathecal treatments. Local levels of TNF-α and PGE2 were immunoenzymatically determined. Incised mice exhibited hypernociception and upregulated expression of Nav1.8 and Nav1.9 in DRG. Antisense oligodeoxynucleotides reduced hypernociception and downregulated Nav1.8 and Nav1.9. TNF-α and COX-2/PGE2 were upregulated in DRG and plantar skin. Inhibition of TNF-α and COX-2 reduced hypernociception, but only TNF-α inhibition downregulated Nav1.8 and Nav1.9. Antagonizing NF-κB and p38 mitogen-activated protein kinase (MAPK), but not ERK or JNK, reduced both hypernociception and hyperexpression of Nav1.8 and Nav1.9. This study proposes the contribution of the TNF-α/p38/NF-κB/Nav1.8 and Nav1.9 pathways to the pathophysiology of the mouse model of incisional pain.

The Compound (E)-2-Cyano-N,3-diphenylacrylamide (JMPR-01): A Potential Drug for Treatment of Inflammatory Diseases.

The compound (E)-2-cyano-N,3-diphenylacrylamide (JMPR-01) was structurally developed using bioisosteric modifications of a hybrid prototype as formed from fragments of indomethacin and paracetamol. Initially, in vitro assays were performed to determine cell viability (in macrophage cultures), and its ability to modulate the synthesis of nitrite and cytokines (IL-1ß and TNFα) in non-cytotoxic concentrations. In vivo, anti-inflammatory activity was explored using the CFA-induced paw edema and zymosan-induced peritonitis models. To investigate possible molecular targets, molecular docking was performed with the following crystallographic structures: LT-A4-H, PDE4B, COX-2, 5-LOX, and iNOS. As results, we observed a significant reduction in the production of nitrite and IL-1ß at all concentrations used, and also for TNFα with JMPR-01 at 50 and 25 µM. The anti-edematogenic activity of JMPR-01 (100 mg/kg) was significant, reducing edema at 2-6 h, similar to the dexamethasone control. In induced peritonitis, JMPR-01 reduced leukocyte migration by 61.8, 68.5, and 90.5% at respective doses of 5, 10, and 50 mg/kg. In silico, JMPR-01 presented satisfactory coupling; mainly with LT-A4-H, PDE4B, and iNOS. These preliminary results demonstrate the strong potential of JMPR-01 to become a drug for the treatment of inflammatory diseases.

Physalis angulata concentrated ethanolic extract suppresses nociception and inflammation by modulating cytokines and prostanoids pathways.

Physalins are seco-steroids with a variety of pharmacological activities already described. In this study the pharmacological properties of a standardized concentrated ethanolic extract from Physalis angulata (CEEPA), rich in physalins B, D, F and G, were studied in models of pain and inflammation in mice. Inflammatory mediators were measured by radioimmunoassay and Real-Time PCR in mice paws after the CFA stimuli. Systemic administration of CEEPA produced antinociceptive effect on the writhing test and formalin test. In the writhing test, physalins B, D, F and G showed that the antinociceptive effect of CEEPA is more potent than that of these purified compounds. In addition, CEEPA reduced the levels of TNF-α, IL-1ß, COX-2 and iNOS mRNA in the CFA-induced paw inflammation. Likewise, CEEPA decreased the TNF-α, IL-1ß and PGE2 paw levels. In conclusion, CEEPA induces antinociceptive and anti-inflammatory effects, with improved pharmacological potency relative to pure physalins, associated to modulation of cytokine and cyclooxygenase pathways.

Mesenchymal stem cells reduce the oxaliplatin-induced sensory neuropathy through the reestablishment of redox homeostasis in the spinal cord.

AIMS: The present study was designed to investigate whether the antinociceptive effect of bone marrow-derived mesenchymal stem/stromal cells (MSC) during oxaliplatin (OXL)-induced sensory neuropathy is related to antioxidant properties. MAIN METHODS: Male mice C57BL/6 were submitted to repeated intravenous administration of OXL (1 mg/kg, 9 administrations). After the establishment of sensory neuropathy, mice were treated with a single intravenous administration of MSC (1 × 106), vehicle or gabapentin. Paw mechanical and thermal nociceptive thresholds were evaluated through von Frey filaments and cold plate test, respectively. Motor performance was evaluated in the rota-rod test. Gene expression profile, cytokine levels, and oxidative stress markers in the spinal cord were evaluated by real-time PCR, ELISA and biochemical assays, respectively. KEY FINDINGS: OXL-treated mice presented behavioral signs of sensory neuropathy, such as mechanical allodynia and thermal hyperalgesia, which were completely reverted by a single administration of MSC. Repeated oral treatment with gabapentin (70 mg/kg) induced only transient antinociception. The IL-1ß and TNF-α spinal levels did not differ between mice with or without sensory neuropathy. MSC increased the levels of anti-inflammatory cytokines, IL-10 and TGF-ß, in the spinal cord of neuropathic mice, in addition to increasing the gene expression of antioxidant factors SOD and Nrf-2. Additionally, nitrite and MDA spinal levels were reduced by the MSC treatment. SIGNIFICANCE: MSC induce reversion of sensory neuropathy induced by OXL possibly by activation of anti-inflammatory and antioxidant pathways, leading to reestablishment of redox homeostasis in the spinal cord.

Phenylpropanoids from Croton velutinus with cytotoxic, trypanocidal and anti-inflammatory activities.

This current study presents the phytochemical analysis of Croton velutinus, describing phenylpropanoids obtained from this species. The fractionation of the roots hexane extract led to the isolation of four new phenylpropanoids derivatives, velutines A-D (1-4) and three known (5-7). Their structures were established based on spectroscopic (1D-2D NMR; HRMS and IR) analysis. Cytotoxic, trypanocidal and anti-inflammatory activities of compounds 1-7 were evaluated. Only compounds 2 and 5 showed cytotoxic activity against cancer cell lines (B16F10, HL-60, HCT116, MCF-7 and HepG2), with IC50 values ranging from 6.8 to 18.3 µM and 11.1 to 18.3 µM, respectively. Compounds 2 and 5 also showed trypanocidal activity against bloodstream trypomastigotes with EC50 values of 9.0 and 9.58 µM, respectively. Finally, the anti-inflammatory potential of these compounds was evaluated on cultures of activated macrophages. All compounds exhibited concentration-dependent suppressive activity on the production of nitrite and IL-1ß by macrophages stimulated with LPS and IFN-γ. These results indicate phenylpropanoids esters (2 and 5) from C. velutinus as promising cytotoxic, trypanocidal and anti-inflammatory candidates that warrants further studies.

IGF-1 overexpression improves mesenchymal stem cell survival and promotes neurological recovery after spinal cord injury.

BACKGROUND: Survival and therapeutic actions of bone marrow-derived mesenchymal stem cells (BMMSCs) can be limited by the hostile microenvironment present during acute spinal cord injury (SCI). Here, we investigated whether BMMSCs overexpressing insulin-like growth factor 1 (IGF-1), a cytokine involved in neural development and injury repair, improved the therapeutic effects of BMMSCs in SCI. METHODS: Using a SCI contusion model in C57Bl/6 mice, we transplanted IGF-1 overexpressing or wild-type BMMSCs into the lesion site following SCI and evaluated cell survival, proliferation, immunomodulation, oxidative stress, myelination, and functional outcomes. RESULTS: BMMSC-IGF1 transplantation was associated with increased cell survival and recruitment of endogenous neural progenitor cells compared to BMMSC- or saline-treated controls. Modulation of gene expression of pro- and anti-inflammatory mediators was observed after BMMSC-IGF1 and compared to saline- and BMMSC-treated mice. Treatment with BMMSC-IGF1 restored spinal cord redox homeostasis by upregulating antioxidant defense genes. BMMSC-IGF1 protected against SCI-induced myelin loss, showing more compact myelin 28 days after SCI. Functional analyses demonstrated significant gains in BMS score and gait analysis in BMMSC-IGF1, compared to BMMSC or saline treatment. CONCLUSIONS: Overexpression of IGF-1 in BMMSC resulted in increased cell survival, immunomodulation, myelination, and functional improvements, suggesting that IGF-1 facilitates the regenerative actions of BMMSC in acute SCI.

Fluorescent Canthin-6-one Alkaloids from Simaba bahiensis: Isolation, Identification, and Cell-Labeling Properties.

Canthin-6-one alkaloids, which are present in plants of the genus Simaba, are natural compounds that are capable of acting as fluorescent probes. However, the chemical composition and fluorescent properties of most species of this genus have not been analyzed. The objective of this study was to characterize the fluorescent properties of an extract of S. bahiensis and identify the chemical entities responsible for these properties. In addition, the cell-labeling properties of the fluorescent dye from A and of the isolated compounds were characterized by confocal fluorescence microscopy and flow cytometry. One quassinoid and three fluorescent alkaloids were isolated from S. bahiensis, all compounds were identified by using NMR spectroscopy and high-resolution mass spectrometry. Staining experiments and HPLC-FL analysis shown that canthin-6-one alkaloids are the main green fluorescent compounds in the analyzed dyes. All compounds evaluated showed a cytoplasmic marker with a residence time of 24 h. The present study is the first to describe the presence of canthin-6-one alkaloids in S. bahiensis, in addition to demonstrating promising cell-labeling properties of fluorescent compounds from S. bahiensis with broad emission wavelengths.

Pain-like behaviors and local mechanisms involved in the nociception experimentally induced by Latrodectus curacaviensis spider venom.

The present study was undertaken to characterize the behavioral manifestations of nociception and the local mechanisms involved with the nociceptive response elicited by Latrodectus curacaviensis venom (LCV) in mice. After the intraplantar LCV inoculation, spontaneous nociception, mechanical and thermal nociceptive thresholds, motor performance, edema and cytokine levels were evaluated using von Frey filaments, hot/cold plate, rota-rod, plethismometer and ELISA, respectively. Analysis of LCV was performed by SDS-PAGE and chromatography. Intraplantar injection of LCV (1-100 ng/paw) induced intense and heat-sensitive spontaneous nociception, mediated by serotonin and bradykinin receptors, TRPV1 channels, as well as by transient local inflammation. LCV (0.1-10 ng/paw) induced mechanical allodynia, which was reduced by the local pretreatment with H1 receptor or TRPV1 antagonists. Corroborating the TRPV1 involvement, in thermal nociception assays, LCV induced a similar response to that of capsaicin, a TRPV1 agonist, facilitating the response to noxious hot stimuli and inhibiting the response to cold noxious stimulation. LCV promoted mast cell degranulation, increased IL-1ß paw levels, but did not produce a relevant edematogenic effect. Analysis of LCV components showed a predominance of high molecular weight proteins. This work provides the first mechanistic hypothesis to explain the local pain induced by LCV, the most frequent clinical symptom of human envenomation.

Nanomolar anti-sickling compounds identified by ligand-based pharmacophore approach.

Adenosine receptors are considered as potential targets for drug development against several diseases. The discovery of subtype 2B adenosine receptors role in erythrocyte sickling process proved its importance to neglected diseases such as sickle cell anemia, which affects approximately 29.000 people around the world, but whose treatment is restricted to just one FDA approved drug (hydroxyurea). In order to widen the therapeutic arsenal available to treat sickle cell anemia patients, it is imperative to identify new lead compounds that modify the sickling course and not just its symptoms. In order to accomplish this goal, ligand-based pharmacophore models that differentiate true ligands from decoys and enlighten the structure-activity relationship of known RA2B antagonists were employed screen the lead-like subset of the ZINC database. Following a chemical diversity analysis, 18 compounds were selected for biological evaluation. Among them, one molecule Z1139491704 (pEC50 = 7.77 ± 0.17) has shown better anti-sickling activity than MRS1754 (pEC50 = 7.63 ± 0.12), a commercial RA2B antagonist. Moreover, these compounds exhibited no cytotoxic effect at low micromolar range on mammalian cells. In conclusion, the sound development of validated ligand-based pharmacophore models proved essential to identify novel chemical scaffolds that might be useful to develop anti-sickling drugs.

Bisphosphonates: Pharmacokinetics, bioavailability, mechanisms of action, clinical applications in children, and effects on tooth development.

Bisphosphonates (BPs) avidly bind to calcium crystals and inhibit osteoclastic bone resorption, making them useful for treatment of skeletal disorders such as osteoporosis, Paget's disease, osteogenesis imperfecta and metastatic bone diseases. BPs therapeutically act by causing toxic effects on osteoclasts or interfering with specific intracellular pathways in those cells. BPs that possess nitrogen in their composition are called nitrogen-containing BPs (NBPs) and include alendronate, pamidronate, risedronate, ibandronate, and zoledronate. Simple BPs or non-NBPs do not have nitrogen in their composition, include etiodronate and clodronate, and were the first to be tested in animals and clinically used. Because BPs may be administered to pregnant women or children during deciduous and permanent teeth development, it is expected that they might disturb tooth eruption and development. A review of current literature on pharmacokinetics, bioavailability, mechanisms of action, and clinical applications of BPs in children, and their effects on tooth eruption and development is presented.